Process for preparing agents containing virus-inactivated vitamin k- dependent plasma components as well as protein c and protein s by membrane chromatography

ABSTRACT

Disclosed is a process for preparing agents containing virus-inactivated vitamin K-dependent plasma components as well as protein C, protein S, factors II, VII, IX and/or X as well as combinations thereof, such as, for example, PPSB preparations, wherein a source containing these components is subjected to appropriate separation procedures, especially by using membrane-chromatographic methods.

[0001] The subject matter of the present invention is a process forpreparing agents containing virus-inactivated vitamin K-dependent plasmacomponents as well as protein C and protein S from a source containingthese components.

[0002] Vitamin K-dependent plasma components such as protein C, proteinS, the factors II, VII, IX and X are constituents which are contained inblood plasma and play an important role in the pathophysiology of theblood clotting cascade. These factors are employed as medicaments in thetherapy of patients exhibiting symptoms caused by a respectivedeficiency in these factors.

[0003] Now, blood plasma is not available in any desired amount as asource for a commercial recovery of the factors. Thus, for both ethicaland economic reasons it must be the goal of any fractionation andisolation of these vitamin K-dependent plasma components as well asprotein C and protein S to ensure a yield as high as possible of eachfactor alone by itself, on the one hand, and at the same time to alsoallow an isolation of each of the other factors, on the other hand. Itis the object of the invention to provide a process capable of reachingsaid goal.

[0004] The object as posed is attained by the process according to theinvention comprising the features set forth in claim 1.

[0005] In the process according to the invention, the use of membranechromatography plays an important role. Josić et al., in Journal ofChromatography, 632 (1993), 1-10, describe the suitability of theheparin affinity chromatography for isolating plasma proteins from theblood clotting cascade. The isolation of antithrombin III and theseparation of factor IX and factor X after a preliminary separation byanion chromatography is described. The anion exchanger chromatographyfor a preliminary separation of factor IX and factor X is effected bypreparative HPLC. The enrichment of samples containing factor IX andfactor X is accomplished in accordance with H. G. J. Brummelhuis, in J.M. Curling (Editor), “Methods of Plasma Protein Fractionation”, AcademicPress, London, Orlando, 1980, p. 117.

[0006] The high performance membrane chromatography of serum and plasmamembrane proteins has already been known from Josic et al., Journal ofChromatography, 590 (1992), 59-76. The separation of serum and plasmamembrane proteins has been described therein. For example, plasmamembrane proteins from liver and kidney tissues were separated.

[0007] Surprisingly it has been shown that valuable vitamin K-dependentplasma ingredients such as protein C, protein S, factors II, VII, IX andX can be obtained in high purity and yield by means of the processdescribed hereinbelow.

[0008] In a first step, the sources containing the respective vitaminK-dependent plasma components as well as protein C and protein S, whichsources are preferably employed from blood plasma in the fresh or thawedcondition, are subjected to a solid phase extraction on anion exchangermaterials. The anion exchanger material may be employed as a particulatematerial in a loose bulk, arranged in membranes or in the form ofcompact disks. The solid phase extraction on polysaccharides which havebeen modified with basic groups and optionally cross-linked ispreferred. More specifically, materials such as polysaccharides modifiedwith diethylaminoethyl groups (DEAE) or quaternary amines can be used.Thus, e.g., materials such as Sephadex® P 50 may be employed.DEAE-modified separating materials, more particularly, are used for theseparation of factor IX. In the solid phase extraction, the source to beextracted is admixed with the material which is present in the solidstate.

[0009] The solid phase extraction is preferably carried out underconditions of low ionic strength. After steps of washing which areoptionally carried out, the effluent is collected and may then besubjected to other work-up steps.

[0010] Thus, in order to separate factor IX, the sample is preferablyfiltered through an appropriately modified membrane. Then the filtratemay optionally be further used, e.g., for the production of albumin.

[0011] The proteins bound to the membrane or the respective solid phasematerial, i.e. factor II, especially factor IX and factor X, aresubsequently eluted under conditions of a higher ionic strength from thesolid phase. When DEAE- or quaternary amine-modified membranes are used,the multiple usability thereof constitutes an advantage over the solidphase extraction by means of the above-mentioned solid phase extractionmaterials, especially the particulate materials.

[0012] The material adsorbed on the solid phase is then desorbed fromthe carrier material by the action of solutions having a higher ionicstrength. Then the ionic strength of the fraction will be adapted to theconditions required for performing the subsequent separation steps bysuitable measures such as dilution or ultrafiltration or diafiltrationor addition of agents increasing the ionic strength.

[0013] This may be followed by an anion exchanger membranechromatography or affinity membrane chromatography using immobilizedsubstances having a high or low molecular weight which have a highaffinity to the vitamin K-dependent plasma components to be isolated aswell as protein C and protein S.

[0014] This step of a chromatographic purification may also be effectedon materials for carrying out a hydrophobic interaction chromatography.

[0015] As the anion exchanger materials, more particularly, membranesmodified with diethylaminoethyl groups or quaternary amines,respectively, are to be taken into consideration also here. Substanceshaving a high affinity to the vitamin K-dependent plasma components aswell as protein C and protein S may be immunoaffinity ligands.Immunoaffinity ligands to be considered are antibodies directed againstthe factors to be isolated. Thus, for example, for the isolation offactor IX, membranes carrying respective immobilized antibodies againstfactor IX can be used. The substances that are not retained by the anionexchanger membrane or immunoaffinity membrane are washed out, optionallycollected and further processed.

[0016] Typical ligands for the hydrophobic chromatography exhibit somegradual changes in hydrophobicity. They include, for example, acyclic oralicyclic aliphatic compounds having, for example, C₁- to C₁₈-alkylchains or aromatic compounds which may also have been modified withpolar protic or polar aprotic ligands such as cyano groups. As thehydrophobic ligands suitable for hydrophobic interaction chromatographythere are to be especially considered propyl, butyl, phenyl groups bymeans of which the carrier material has been modified and similarligands exhibiting some gradual change in hydrophobicity. The gradualchange in hydrophobicity may also be effected by polar groups. Thus,more particularly, 2-hydroxyaminoalkyl groups, such as2-hydroxyaminopropyl groups, are suitable as hydrophobic ligands for theisolation of factor IX.

[0017] The next step comprises a further fractionation of the adsorbedplasma components by stepwise elution upon a change in the ionicstrength and/or the pH value either with solvent systems of higher ionicstrength solvent systems having different polarities or solvent systemsreversing the affinity between the immuno-affinity ligand and thesubstrate. The procedures mentioned above may then be adopted to adjustthe ionic strength of the fraction eluting from the membrane to theconditions of further purification.

[0018] The optional step f) of claim 1 comprises an affinity membranechromatography. The affinity membrane chromatography within the scope ofthe process according to the invention is understood to also includehydrophobic chromatography. Appropriate ligands have already beencharacterized hereinabove.

[0019] The affinity membrane chromatography within the scope of theprocess according to the invention also relates to chromatographicprocedures wherein membranes modified with immunoaffinity ligands areused. Herein, more specifically, monoclonal antibodies against thevitamin K-dependent plasma components as well as protein C and protein Sare used, which monoclonal antibodies have been immobilized on themembrane.

[0020] The operations of the chromatographic separation of the vitaminK-dependent plasma components as well as protein C and protein S in thesample may be carried out with a use of substrate materials modifiedwith ion exchanger groups, especially anion exchangers, on the one hand,or with a use of materials modified with immunoaffinity ligands, on theother hand.

[0021] In a very advantageous mode said chromatographic materials arearranged in membranes. Preferably, the membranes consist of a substratematerial such as a modified cellulose or a synthetic fiber. Morespecifically, membranes as well as compact disks made of porouspolyglycidyl methacrylates and/or of other porous hydrophilic polymershaving a similar structure, such as a hydrophilized polystyrene, aresuitable.

[0022] A membrane suitable for the separation consists of a stack thinporous films made of cellulose of synthetic fibers in the first case,while it consists of compact disks made from silicagel or polymercarriers in the second case. The substrate materials of said membranesof disks have been provided with the appropriate anion exchanger groupsor immunoaffinity ligands. The ion exchanger groups, more particularly,may be anion exchanger groups such as quaternary ammonium compounds ordiethylaminoethyl (DEAE) groups. The cation exchangers, basically, maybe weakly or strongly acidic cation exchangers such as materialsmodified with sulfonic acid or phosphoric acid groups.

[0023] The ion exchanger groups may or may not have been bonded to thefiber of the substrate material through a so-called spacer. Materialsprovided with spacers are also called tentacle materials. Suitablespacers and ligands have been specified in DE 42 04 694. A glucsoaminemoiety, for example, may also serve as a spacer. Anion exchanger groupssuch as DEAE or quaternary ammonium compounds may also have been bondedto the membranes made of porous polyglycidyl methacrylate or the othermaterials mentioned. The anion exchanger groups are bonded eitherdirectly to the material forming the membrane or also through a spacer,e.g. a glucsoamine moiety.

[0024] In another embodiment of the process according to the invention,an affinity membrane chromatography is used which utilizes immobilizedlow or high molecular weight substances having a high affinity to thevitamin K-dependent plasma components as well as protein C and proteinS, which preferably are of human or murine origin.

[0025] The substances possessing affinity to the vitamin K-dependentplasma components, factors II, VII, IX or X as well as protein C andprotein S are immobilized on the carrier by means of chemically activegroups. It is preferred that the active group will not directly attackthe carrier material, but will attack at the end of a spacer. Theimmobilization of the substances having affinity to the factors iseffected by bonding same to active grops such as tosyl, tresyl,hydrazide and others. Appropriate procedures have been known from T. M.Phillips, “Affinity Chromatography”, in “Chromatography” (E. Heftmann,Ed.), 5th edition, Elsevier, Amsterdam 1992.

[0026] The antibodies may also be preliminarily adsorbed on membranesbearing protein A or protein G ligands. The elution of the antibodies(bleeding of the column) may be prevented by a subsequent covalentcross-linking of the column. For cross-linking the antibodies to proteinA or protein G membranes, a process similar to that using loose carriersmay be employed. The advantage of immobilizing on protein A or protein Gconsists of that the antibodies are exclusively immobilized on theconstant segment of the molecule (F_(c)). Hence, the antigen-bindingportion (F_(ab)) remains free and is not inhibited in its interactionwith the respective factors.

[0027] The virus-inactivation is effected by treating the fractionobtained after a chromatographic purification with detergents such asionic and/or non-ionic surfactants, e.g. in the presence of di- ortri-alkylated phosphate compounds, such as, e.g., tri-n-butyl phosphateaccording to the method described in EP 0 131 740 A1. Basically, thevirus-inactivation may also be carried out prior to firstchromatographic step. It is preferred that Triton® X-100 Tween/TNBP(tri-n-butyl phosphate) are used for the virus-inactivation. Goodresults are also obtained with sodium cholate/TNBP. Preferably,quantities of up to 15% by weight of the detergent are used.

[0028] However, the virus-inactivation may also be effected by means ofa heat treatment. In this procedure, after a first membranechromatography, the vitamin K-dependent plasma components as well asprotein C and protein S are subjected to a step of pasteurization. Anappropriate process is proposed in the German patent application P 43 18435.9. Therein, fractions enriched with factor VIII are brought intocontact with di- or trialkyl phosphates and optionally wetting agents inthe presence of stabilizers such as sugars, amino acids, bivalentcations and/or heparin and, at the same time or subsequently, aretreated at some elevated temperature within the range of from 55° C. to70° C. for a period of from 5 hours to 30 hours. If so desired, afiltration for removing viruses may also be carried out.

[0029] It may be advantageous to combine the two methods ofvirus-inactivation, treatment with detergents and heat as well asfiltration.

[0030] In the isolation of factor IX, the pasteurization step ispreferably carried out subsequently to step f) of claim 1. This may befollowed by another membrane chromatography for removing the chemicalsused in said step. It is preferred that the stabilizers added areremoved by means of membrane modified with DEAE or quaternary ammoniumcompounds positioned on the surface of the chromatographic carriermaterial through a spacer. It is also possible to position thecorresponding ligands on the surface of the chromatographic carriermaterial without using a spacer.

[0031] Under the conditions chosen, the stabilizers are not retarded bythis anion exchanger material, whereas the factors are adsorbed on thechromatography material.

[0032] The stabilizers which in general consist of lower molecularweight substances may also be removed by ultra- or diafiltration. Theresulting fractions accumulated with vitamin K-dependent plasmacomponents such as protein C and protein S are then concentrated, if sodesired. The concentration methods offering themselves are proceduresinvolving the removal of the solvent, usually water, under mildconditions. They include, more specifically, procedures wherein thesolvent is removed under reduced pressure, such as, for example,lyophilization (freeze drying) or spray drying.

[0033] The utilization of membrane chromatograpy in the processaccording to the invention, more particularly, implies the advantage ofthat the chromatographic separation may be carried out considerablyfaster. Furthermore, the membranes to be utilized can be re-used amultiplicity of times. In contrast thereto, if the solid phase materialaccording to prior art is used, this material cannot be re-used, butwill already have to be disposed of if used once.

[0034] The process according to the invention is illustrated in greaterdetail by way of the isolation of factor IX.

EXAMPLE 1

[0035] Thawed blood plasma is subjected to a solid phase extraction withSephadex® P 50. After the elution of the substance adsorbed on the solidphase, the resulting fraction is adjusted to have an ionic strengthcorresponding to from 10 to 20 mM of sodium citrate at pH 7.4 and issubjected to a membrane chromatography in a DEAE QuickDisk (diameter of25 mm; thickness of 3 mm). The pressure is approximately 3 bar. Then,chromatography is carried out at a flow rate of 5 ml/min. The pooledfraction contains a mixture of factor II and factor VII and may besubjected to a further work-up. The peak eluting then from the columncontains a mixture of factor IX and factor X. Membrane chromatography iscarried out by employing a gradient which changes from said initialbuffer solution towards a buffer solution comprising 1 M of NaCl as wellas from 10 to 20 mM of sodium citrate of pH 7.4 as buffer B. If a stronganion exchanger having a high surface occupation of ligands will beused, a higher capacity will be achieved. Some limiting factor isconstituted by the selectivity of the material. The course of theelution gradient will depend on the degree of surface occupation ofligands on the carrier.

[0036] The mixture comprising factor IX and factor X is then subjectedto further chromatography. The load capacity of the membranechromatographic material employed is equal to or greater that of thematerial in the form of particles, both based on the amount of material.

EXAMPLE 2

[0037] The fraction recovered according to Example 1 and containing orIX/X is treated as per Journal of Chromatography, 632 (1993), 1-10. Thefraction containing factor IX/X as obtained according to Example 1 issubjected to a heparin affinity membrane chromatography on a compactdisk. After application from a buffer having a relatively low ionicstrength, the material is rinsed with a buffer having a ionic strengthof about 500 mOsm. Then, factor IX is eluted by using a gradient whichstarts at about 500 mOsm and increases up to about 1,000 mOsm. Thefactor IX eluted thereby has a high purity. The rate of the recovery offactor IX, starting from the first work-up step, is about 87%.

1. A process for preparing agents containing virus-inactivatd vitamink-dependent plasma components as well as protein c, protein s, factorsII, VII, IX and/or X as well as combinations thereof, such as, forexample, PPSB preparations, wherein a source containing these componentsis subjected to the following process steps: a) Solid phase extractionof the sources containing the components to be separated on anionexchanger materials in loose bulk, on membranes and/or on compact disksunder conditions of relatively low ionic strength, removal of theresulting effluent and, if desired, supplying the effluent to furtherwork-up procedures for the recovery of other materials; b) elution ofthe material adsorbed on the solid phase; c) optionally, adjustment ofthe ionic strength and/or of the pH value of the fraction containing theeluate to the condition of the following purification step; d)virus-inactivation by means of ionic and/or non-ionic detergents in thepresence of di- or trialkyl phosphate compounds such as tri-n-butylphosphate and optionally a heat treatment; e) a ion exchange membranechromatography, affinity membrane chromatography with immobilized highor low molecular weight substances possessing a high affinity to vitaminK-dependent plasma components as well as protein C and protein S orhydrophobic interaction chromatography; f) fractionation of individualcomponents by stepwise elution by changing the ionic strength, thepolarity and/or the pH value; g) optionally membrane affinitychromatography; h) optionally collecting the eluate that contains theproducts which have not already been removed, and optionally the furtherfractionation thereof by repeating step f), wherein appropriate affinitymaterials are used; i) elution of the substances bound to the affinitymaterial under conditions reversing the affinity bond, followed byconcentration of the eluate, optionally with reducing the amount of theagent used for the elution from the affinity material; or the virusinactivation tades place after step f) followed by a membranechromatography step and optionally a heat treatment and/or a filtrationfor the removal of viruses takes place at a suitable stage.
 2. Theprocess according to claim 1, wherein the source containing vitaminK-dependent plasma components such as protein C and protein S is bloodplasma in fresh or thawed condition.
 3. The process according to claims1 and/or 2, wherein the solid phase extraction is effected by means of across-linked polysaccharide modified with basic groups, such asSephadex® or by means of anion exchanger membranes appropriatelymodified.
 4. The process according to at least one of claims 1 to 3,wherein the membrane chrmatography is carried out by using membraneswhich have been modified with DEAE or quaternary amines, or by usingmembranes which have been modified by low or high molecular weightaffinity ligands capable of specifically binding to the desired plasmacomponents (membrane affinity chromatography).
 5. The process accordingto at least one of claims 1 to 4, wherein the affinity chromatography isa heparin membrane affinity chromatography, a membrane immunoaffinitychromatography with immobilized antibodies against the substance to beisolated or a membrane affinity chromatography using membranes withhydrophobic ligands.
 6. The process according to claim 5, wherein thehydrophobic ligands for modifying the chromatographic carrier are2-hydroxyaminoalkyl groups such as 2-hydroxyaminopropyl and/orhydrophobic ligands such as propyl, butyl, phenyl groups and othersimilar ligands exhibiting some gradual change in hydrophobicity.
 7. Theprocess according to at least one of claims 1 to 6, wherein theadjustment of the ionic strength after the chromatographic steps iseffected by dilution or desalting procedures such as dia- orultrafiltration or by the addition of agents increasing the ionicstrength.
 8. The process according to at least one of claims 1 to 7,wherein the virus-inactivation is effected by heating the fraction atfrom 55° C. to 70° C. for a period of from 5 to 30 hours in the presenceof stabilizers such as sugar, amino acids, bivalent cations and/orheparin.
 9. The process according to claim 8, wherein the stabilizersare removed by desalting procedures such as diafiltration orultrafiltration, by heparin affinity chromatography or anion exchangerchromatography or on carriers modified with DEAE or quaternary ammoniumcompounds or carriers modified with hydrophobic ligands.
 10. The processaccording to at least one of claims 1 to 9, wherein the chromatographymaterial is a particulate material arranged as loose bulk, a materialembedded in membranes and/or compact disks made of the respectivematerials.
 11. The process according to at least one of claims 1 to 10,wherein the fractions obtained are concentrated by lyophilization orspray drying.